CN106305425A - Method for producing purple pigment from callus tissue of panax notoginseng - Google Patents
Method for producing purple pigment from callus tissue of panax notoginseng Download PDFInfo
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- CN106305425A CN106305425A CN201610726125.1A CN201610726125A CN106305425A CN 106305425 A CN106305425 A CN 106305425A CN 201610726125 A CN201610726125 A CN 201610726125A CN 106305425 A CN106305425 A CN 106305425A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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Abstract
The invention discloses a method for producing purple pigment from the callus tissue of panax notoginseng, the method comprises the steps that the tender stem, leaf, flower or anther of panax notoginseng plant are used as explants, the explants are subjected to sterilization, inoculation and induction culture to produce the callus tissue; the callus tissue is subjected to secondary sterilization, secondary inoculation and secondary culture to obtain a passage callus tissue, and purple pigment is extracted from the passage callus tissue. Through controlling the conditions, such as illumination time, culture medium, humidness and temperature, the callus tissue rich in purple pigment and high in proliferation rate are cultured. The content of the panax notoginseng purple pigment is effectively improved through the illumination control; TDZ is added in the culture medium and matched with other plant hormones, so that the induction rate of the callus tissue of panax notoginseng is obviously improved, and the formation of purple pigment is facilitated.
Description
Technical field
The invention belongs to biological field, be specifically related to a kind of method utilizing Radix Notoginseng callus to produce purpurin.
Background technology
Radix Notoginseng is araliaceae ginseng plant, has the good reputations such as " Radix Stephaniae Sinicae (Radix Stephaniae Dielsianae) ", " southern part of the country SHENCAO ", " king in ginseng ", is Yunnan
Distinctive rare Chinese medicine, a bright jewel in Ye Shi China Chinese medicine.Radix Notoginseng purpurin is that one is present in Radix Notoginseng and plants
Strain stem, leaf, Hua Jigen Cell sap in compound, make plant present the different colours such as red, purplish red, blue.
Radix Notoginseng purpurin be in the nature anthocyanin, anthocyanin is flavonoid--energy in the class material based on flavone core
Present a compounds of group of redness.Owing to it has the functional of uniqueness, and it is applied to removing interior free yl, propagation leaf Huang
Element, antitumor, anticancer, antiinflammatory, anti-lipid peroxidation and platelet aggregation, prevention diabetes, lose weight, protect vision etc..Flower
Color glycosides is as a kind of natural pigment, safe and nontoxic, and has many health cares to human body, has been applied to food, health care
The industries such as product, cosmetics, medicine.
But, utilizing and the pertinent literature developed and report at present and independent of Radix Notoginseng purpurin.
Summary of the invention
It is an object of the invention to provide and a kind of turn out the Radix Notoginseng callus rich in purpurin by tissue culture's means,
And utilize it to extract purpurin.
The technical scheme that the present invention provides:
A kind of method utilizing Radix Notoginseng callus to produce purpurin, makees with stem, leaf, flower or the flower pesticide that Radix Notoginseng plant children is tender
For outer implant, produce callus through sterilizing, inoculation, inducing culture, callus is carried out two-stage sterilization, inoculation and subculture
Cultivation obtains passing on callus, and extracts described purpurin callus from passing on；
Illumination in wherein said inducing culture controls to be 4～8 hours illumination conditions and dark condition friendship in 4～8 hours
For carrying out；
The illumination of described successive transfer culture controls to be 10～15 hours illumination conditions and 10～15 hours dark conditions replace into
OK.
Further, the culture medium of described induction of callus is: MS+0.1～2mg/L 2,4-D+0.1～2mg/
L BA+0.002～1mg/L TDZ+30～60g/L sucrose (that is: add 0.1～2mg 2,4-in the MS culture medium of each liter
D, 0.1～2mg BA, 0.002～1mg TDZ and 30～60g sucrose)；Or MS+0.1～2mg/L NAA+0.1～2mg/L BA+
0.002～1mg/L TDZ+30～60g/L sucrose (that is: add in the MS culture medium of each liter 0.1～2mg NAA, 0.1～
2mg BA, 0.002～1mg TDZ and 30～60g sucrose).
Described MS is MS culture medium, and NAA is naphthalene acetic acid, and 2,4-D is 2,4-dichlorphenoxyacetic acid, and BA is that benzyl amino gland is fast
Purine, TDZ is Thidiazuron.
Further, the culture medium of described callus successive transfer culture is: MS+0.1～2mg/L NAA+0.1～2mg/L
BA+0.002～4mg/L TDZ (that is: in the MS culture medium of each liter add 0.1～2mg NAA, 0.1～2mg BA and
0.002～1mg TDZ).
Further, the intensity of illumination of described inducing culture is 1500 2000lx.
Further, the intensity of illumination of described successive transfer culture is 1000 1500lx.
Further, the size of described callus is diameter 1～2cm.
Further, the extracting method of described purpurin includes solvent extraction, supersound extraction, supercritical fluid extraction, micro-
One or more in ripple extraction.
Further, the purification process to described purpurin is also included.
Further, described purification process includes extraction, recrystallization, column chromatography for separation, and macroporous resin separates, ion tree
One or more during fat separates combine.
Beneficial effects of the present invention:
The present invention, by controlling the conditions such as light application time, culture medium, humidity, temperature, turns out rich in purpurin, induction
The callus that rate is high.The present invention is shortened by the method for illumination and dark alt time and effectively raises Radix Notoginseng purpurin
Content, and add TDZ and the collocation with other plant hormone in the medium, make the inductivity of Radix Notoginseng callus substantially carry
Height, and the formation of beneficially purpurin.
The present invention also carries out inducing culture to callus simultaneously, makes the callus induced avoid differentiation, is formed big
The callus of amount propagation, and to the culture medium of successive transfer culture and environmental Kuznets Curves, to condition controls such as light application time, humidity, temperature
System, turns out rich in purpurin, callus that the rate of increase is high.The Radix Notoginseng callus purpurin content of inducing culture of the present invention
Height, inductivity is high, and the rate of increase is high.
The present invention utilizes the advantage that tissue culture propagating efficiency is high, growth cycle is short, sets up and is suitable for Radix Notoginseng purpurin wound healing
The condition of tissue culture of tissue growth, thus provide the callus rich in purpurin of substantial amounts of high-quality, for from Radix Notoginseng callus
Middle extraction purpurin provides material base, beneficially automatization, large-scale production, improves production efficiency.
Detailed description of the invention
Embodiment 1
A kind of method utilizing Radix Notoginseng callus to produce purpurin, comprises the following steps:
Choose the Radix Notoginseng plant tender stem of children of health, leaf, flower as outer implant, rinse 5 times with clear water, first with 70% wine
Essence sterilizing 10s, after aseptic water washing 3 times, then will rinse after outer implant be placed in the mercuric chloride that concentration is 0.1%, immersion is gone out
Bacterium, sterile deionized water rinses 6 times, standby.
0.5cm will be cut into through the stem of sterilizing, leaf, flower in gnotobasis2Block outer implant；In aseptic condition
Under, open tissue culture bottle at alcohol burner flame vicinity, the outer implant cut once, is inoculated into culture medium by bottleneck calcination on flame
On, every bottle of culture medium inoculated 3～5 pieces of outer implant.
The culture medium of MS+0.1mg/L 2,4-D+0.1mg/L BA+0.002mg/L TDZ+30g/L sucrose lures
Leading cultivation, cultivation cycle is 45 weeks.
Wherein, illumination control be 4 hours illumination conditions and 4 hours dark conditions alternately, intensity of illumination is 1500lx；
Temperature: 22 DEG C；Relative air humidity: 50%.
Choose the Radix Notoginseng callus growing to diameter 1cm, in gnotobasis, Radix Notoginseng callus is divided into
0.5mm2Square fritter, under sterile conditions, open tissue culture bottle at alcohol burner flame vicinity, bottleneck is calcination one on flame
Under, the callus cut is inoculated on subculture medium,
Wherein, the culture medium of successive transfer culture is MS+0.1mg/L NAA+0.1mg/L BA+0.002mg/L TDZ, cultivates 4
In week, obtain passing on callus.
Wherein, illumination controls to be illumination in 10 hours, 10 hours dark, and alternately, the light intensity that illumination controls is
1000lx.The temperature of successive transfer culture is 22 DEG C；Relative air humidity: 50%.
The callus organic solvent ethanol that passes on cultivation obtained carries out solvent extraction and supersound extraction, and extracts
Take, recrystallization purifying obtains purpurin.
Embodiment 2
A kind of method utilizing Radix Notoginseng callus to produce purpurin, comprises the following steps:
Choose the Radix Notoginseng plant tender stem of children of health, leaf, flower as outer implant, rinse 6 times with clear water, first with 70% wine
Essence sterilizing 10s, after aseptic water washing 3 times, then will rinse after outer implant be placed in the mercuric chloride that concentration is 0.1%, immersion is gone out
Bacterium, sterile deionized water rinses 8 times, standby.
0.5cm will be cut into through the stem of sterilizing, leaf, flower in gnotobasis2Block outer implant；In aseptic condition
Under, open tissue culture bottle at alcohol burner flame vicinity, the outer implant cut once, is inoculated into culture medium by bottleneck calcination on flame
On, 5 pieces of outer implant of every bottle of culture medium inoculated.
Inducing culture therein is MS+2mg/L 2,4-D+2mg/L BA+1mg/L TDZ+60g/L sucrose.
Wherein, illumination control be 8 hours illumination conditions and 8 hours dark conditions alternately, intensity of illumination is 2000lx；
Temperature: 25 DEG C；Relative air humidity: 80%；Cultivation cycle is 5 weeks.
Choose the Radix Notoginseng callus growing to diameter 1-2cm, in gnotobasis, Radix Notoginseng callus is divided into
0.5mm2Square fritter, under sterile conditions, open tissue culture bottle at alcohol burner flame vicinity, bottleneck is calcination one on flame
Under, the callus cut is inoculated on subculture medium,
Wherein, the culture medium of successive transfer culture is MS+2mg/L NAA+2mg/L BA+4mg/L TDZ, cultivates 5 weeks.
Wherein, illumination controls to be illumination in 15 hours, 15 hours dark, and alternately, the light intensity that illumination controls is
1500lx.The temperature of successive transfer culture is 25 DEG C；Relative air humidity: 80%.
The callus that passes on cultivation obtained carries out supersound extraction, and the method purification used column chromatography obtains purple
Element.
Embodiment 3
A kind of method utilizing Radix Notoginseng callus to produce purpurin, comprises the following steps:
Choose the flower pesticide of Radix Notoginseng of health as outer implant, flower of Radix Notoginseng clear water rinsed 5 times, first with 70% ethanol sterilizing
10s, then will rinse after outer implant be placed in the mercuric chloride that concentration is 0.1%, soak sterilizing, sterile deionized water rinse 6 times, standby
With.
Aseptic filter paper strips flower pesticide；Under sterile conditions, opening tissue culture bottle at alcohol burner flame vicinity, bottleneck exists
On flame, calcination is once, is inoculated in tissue culture bottle by flower pesticide, 15 every bottle；
At MS+0.1mg/L NAA+0.1mg/L BA+0.002mg/L TDZ+30g/L sucrose, culture medium in lure
Lead cultivation.
Wherein, illumination control be 6 hours illumination conditions and 6 hours dark conditions alternately, intensity of illumination is 1600lx；
Temperature: 22 DEG C；Relative air humidity: 50%, cultivation cycle is 6 weeks.
Choose the Radix Notoginseng callus growing to diameter 2cm, in gnotobasis, Radix Notoginseng callus is divided into
0.5mm2Square fritter, under sterile conditions, open tissue culture bottle at alcohol burner flame vicinity, bottleneck is calcination one on flame
Under, the callus cut is inoculated on subculture medium,
Wherein, the culture medium of successive transfer culture is MS+1mg/L NAA+1.2mg/L BA+0.08mg/L TDZ, cultivates 4 weeks.
Wherein, illumination controls to be illumination in 12 hours, 12 hours dark, and alternately, the light intensity that illumination controls is
1200lx.The temperature of successive transfer culture is 23 DEG C；Relative air humidity: 60%.
The callus that passes on cultivation obtained carries out supercritical fluid extraction method extraction, and with macroporous resin and post layer
The method purification that analysis separates obtains purpurin.
Embodiment 4
A kind of method utilizing Radix Notoginseng callus to produce purpurin, comprises the following steps:
Choose the flower pesticide of Radix Notoginseng of health as outer implant, flower of Radix Notoginseng clear water rinsed 6 times, first with 70% ethanol sterilizing
10s, then will rinse after outer implant be placed in the mercuric chloride that concentration is 0.1%, soak sterilizing, sterile deionized water rinse 8 times, standby
With.
Aseptic filter paper strips flower pesticide；Under sterile conditions, opening tissue culture bottle at alcohol burner flame vicinity, bottleneck exists
On flame, calcination is once, is inoculated in tissue culture bottle by flower pesticide, 20 every bottle；
Inducing culture is carried out in the culture medium of MS+2mg/L NAA+2mg/L BA+1mg/L TDZ+60g/L sucrose.
Wherein, illumination control be 5 hours illumination conditions and 5 hours dark conditions alternately, intensity of illumination is 1700lx；
Temperature: 25 DEG C；Relative air humidity: 80%, cultivation cycle is 7 weeks.
Choose the Radix Notoginseng callus growing to diameter 2cm, in gnotobasis, Radix Notoginseng callus is divided into
0.5mm2Square fritter, under sterile conditions, open tissue culture bottle at alcohol burner flame vicinity, bottleneck is calcination one on flame
Under, the callus cut is inoculated on subculture medium,
Wherein, the culture medium of successive transfer culture is MS+0.8mg/L NAA+0.5mg/L BA+2mg/L TDZ, cultivates 4 weeks.
Wherein, illumination controls to be illumination in 13 hours, 13 hours dark, and alternately, the light intensity that illumination controls is
1400lx.The temperature of successive transfer culture is 23 DEG C；Relative air humidity: 60%.
The callus that passes on cultivation obtained carries out microwave method extraction, and purifies by the method for ion exchange resin and obtain purple
Pigment.
The inductivity of Radix Notoginseng callus and the impact of purpurin content are tested by different phytohormone:
Choose 4 groups of tender stems of healthy Radix Notoginseng plant children as outer implant, through identical sterilization treatment, be inoculated in respectively
4 groups of inducing cultures: (1) MS+0.1mg/L 2,4-D+0.1mg/L BA+30g/L sucrose, (2) MS+0.1mg/L NAA+
0.1mg/L BA+30g/L sucrose, (3) MS+0.1mg/L 2,4-D+0.1mg/L BA+0.1mg/L TDZ+30g/L sucrose, (4)
MS+0.1mg/L NAA+0.1mg/L BA+0.1mg/L TDZ+30g/L sucrose.
Cultivate in the environment of identical, wherein illumination control be 8 hours illumination conditions and 8 hours dark conditions alternately
Carrying out, intensity of illumination is 2000lx；Temperature: 25 DEG C；Relative air humidity: 80%；To its inductivity and purpurin after cultivating 5 weeks
Content test.
Table 1, different phytohormone are to the inductivity of Radix Notoginseng callus and purpurin content
| Numbering | 2,4-D | NAA | BA | TDZ | Inductivity | Purpurin content |
| 1 | 0.1 | 0 | 0.1 | 0 | 8% | 1% |
| 2 | 0 | 0.1 | 0.1 | 0 | 6% | 2% |
| 3 | 0.1 | 0 | 0.1 | 0.1 | 48% | 18% |
| 4 | 0 | 0.1 | 0.1 | 0.1 | 51% | 19% |
From the results shown in Table 1, the interpolation of TDZ can significantly improve the inductivity of the callus induction of Radix Notoginseng, has
It is beneficial to the propagation of callus and rich in the induction of purpurin callus so that it is the content of purpurin significantly improves.
The successive transfer culture rate of increase of Radix Notoginseng callus and the impact of purpurin content are tested by different phytohormone:
Choose 3 groups of identical Radix Notoginseng calluss and carry out successive transfer culture, in gnotobasis, Radix Notoginseng callus is split
Become 0.5mm2Square fritter, under sterile conditions, open tissue culture bottle at alcohol burner flame vicinity, bottleneck is calcination on flame
Once, respectively the callus cut is inoculated on 3 groups of different subculture mediums,
Wherein, subculture medium is respectively as follows: (1) MS+0.1mg/L TDZ, (2) MS+0.1mg/L NAA+0.1mg/L BA
And (3) MS+0.1mg/L NAA+0.1mg/L BA+0.1mg/L TDZ.
Cultivating in the environment of identical, wherein illumination controls to be 12 hours illumination conditions and dark condition friendship in 12 hours
For carrying out, intensity of illumination is 1000lx；Temperature: 25 DEG C；Relative air humidity: 80%；To its rate of increase and purple after cultivating 5 weeks
The content of element is tested.
Table 2, different phytohormone are to the rate of increase of the successive transfer culture of Radix Notoginseng callus and purpurin content
| Numbering | NAA | BA | TDZ | The rate of increase | Purpurin content |
| 1 | 0 | 0 | 0.1 | 12% | 8% |
| 2 | 0.1 | 0.1 | 0 | 42% | 1% |
| 3 | 0.1 | 0.1 | 0.1 | 72% | 19% |
From the results shown in Table 2, the interpolation of TDZ can significantly facilitate the successive transfer culture of the callus of Radix Notoginseng, energy
Significantly improve the content of purpurin in the rate of increase of callus and callus, it is thus achieved that positive controls for high proliferation rates and purpurin content are high
Callus.The synergism of TDZ Yu NAA and BA is conducive to propagation and the growth of callus, and the generation of purpurin.
The inductivity of callus and the impact of purpurin content are tested by different illumination conditions:
Choose 6 groups of tender stems of healthy Radix Notoginseng plant children as outer implant, through identical sterilization treatment, be inoculated in identical
Inducing culture MS+0.1mg/L 2,4-D+0.1mg/L BA+0.1mg/L TDZ+30g/L sucrose in.
It is carried out different photo-irradiation treatment, and respectively (1) 4 hour illumination condition and 4 hours dark conditions are alternately,
(2) 6 hours illumination conditions and 6 hours dark conditions alternately, (3) 8 hours illumination conditions and 8 hours dark conditions replace into
OK, (4) 12 hours illumination conditions and 12 hours dark conditions alternately, (5) 24 hours illumination conditions and 24 hours dark bars
Alternately, (6) 36 hours illumination conditions and 36 hours dark conditions are alternately for part.
The intensity of illumination of 6 groups is 2000lx；Temperature: 25 DEG C；Relative air humidity: 80%；To its inductivity after cultivating 5 weeks
And the content of purpurin tests.
Table 3, different illumination conditions is to the inductivity of Radix Notoginseng callus and purpurin content
| Illumination alt time | 4 | 6 | 8 | 12 | 24 | 36 |
| Inductivity | 32% | 48% | 39% | 24% | 20% | 18% |
| Purpurin content | 14% | 18% | 12% | 5% | 1% | 1% |
From the results shown in Table 3, illumination is used can be effectively improved Radix Notoginseng wound healing group with dark illumination condition alternately
Knit the formation of middle purpurin, wherein shorten illumination and be directly proportional to the content of purpurin in callus to dark alt time.
The rate of increase of the successive transfer culture of callus and the impact of purpurin content are tested by different illumination conditions:
Choose 6 groups of identical Radix Notoginseng calluss and carry out successive transfer culture, in gnotobasis, Radix Notoginseng callus is split
Become 0.5mm2Square fritter, under sterile conditions, open tissue culture bottle at alcohol burner flame vicinity, bottleneck is calcination on flame
Once, the callus cut is inoculated into the subculture medium of MS+0.1mg/L NAA+0.1mg/L BA+0.1mg/L TDZ
On,
It is carried out different photo-irradiation treatment, and respectively (1) 5 hour illumination condition and 5 hours dark conditions are alternately,
Alternately, (3) 12 hours illumination conditions and 12 hours dark conditions are handed over for (2) 10 hours illumination conditions and 10 hours dark conditions
For carrying out, (4) 15 hours illumination conditions and 15 hours dark conditions alternately, (5) 20 hours illumination conditions and 20 hours black
Alternately, (6) 30 hours illumination conditions and 30 hours dark conditions are alternately for dark condition.
The intensity of illumination of 6 groups is 1000lx；Temperature: 25 DEG C；Relative air humidity: 80%；To its rate of increase after cultivating 5 weeks
And the content of purpurin tests.
Table 4, different illumination conditions is to the rate of increase of the successive transfer culture of Radix Notoginseng callus and purpurin content
| Illumination alt time | 5 | 10 | 12 | 15 | 20 | 30 |
| The rate of increase | 36% | 48% | 70% | 62% | 36% | 30% |
| Purpurin content | 8% | 18% | 19% | 13% | 5% | 3% |
From the results shown in Table 4, illumination and dark illumination condition alternately and purple in Radix Notoginseng callus are used
Element be formed with impact, wherein 12 hours illumination conditions and 12 hours dark conditions are most beneficial for the formation of purpurin, purpurin
Content the highest, also improve the rate of increase of Radix Notoginseng callus successive transfer culture simultaneously.
The inductivity of callus and the impact of purpurin content are tested by different illumination intensity:
Choose 5 groups of tender stems of healthy Radix Notoginseng plant children as outer implant, through identical sterilization treatment, be inoculated in identical
Inducing culture in: MS+0.1mg/L 2,4-D+0.1mg/L BA+0.1mg/L TDZ+30g/L sucrose, be placed in temperature: 25
℃；Relative air humidity: 80%；In 6 hours illumination conditions and 6 hours dark conditions environment alternately,.
Wherein, the intensity of illumination of 5 groups is respectively 800lx, 1500lx, 2000lx, 2500lx；After cultivating 5 weeks, it is induced
The content of rate and purpurin is tested.
Table 5, different illumination intensity is to the inductivity of Radix Notoginseng callus and purpurin content
| Intensity of illumination | 800 | 1500 | 2000 | 2500 | 3000 |
| Inductivity | 9% | 48% | 51% | 24% | 12% |
| Purpurin content | 3% | 18% | 19% | 2% | 1% |
From the results shown in Table 5, intensity of illumination is 1500lx, is conducive to improving Radix Notoginseng wound healing under conditions of 2000lx
The formation of purpurin in tissue.
The rate of increase of the successive transfer culture of callus and the impact of purpurin content are tested by different illumination intensity:
Choose 5 groups of identical Radix Notoginseng calluss and carry out successive transfer culture, in gnotobasis, Radix Notoginseng callus is split
Become 0.5mm2Square fritter, under sterile conditions, open tissue culture bottle at alcohol burner flame vicinity, bottleneck is calcination on flame
Once, the callus cut is inoculated into the subculture medium of MS+0.1mg/L NAA+0.1mg/L BA+0.1mg/L TDZ
On,
Wherein, the intensity of illumination of 5 groups is respectively 800lx, 1000lx, 1500lx, 2000lx, 2500lx；After cultivating 5 weeks right
The content of its rate of increase and purpurin is tested.
Table 6, different illumination intensity is to the rate of increase of the successive transfer culture of Radix Notoginseng callus and purpurin content
| Intensity of illumination | 800 | 1000 | 1500 | 2000 | 2500 | 3000 |
| The rate of increase | 52% | 70% | 72% | 42% | 32% | 12% |
| Purpurin content | 3% | 18% | 19% | 5% | 2% | 0.5% |
From the results shown in Table 6, being formed significantly of purpurin in different illumination intensity and Radix Notoginseng callus
Impact, wherein the intensity of illumination of 1000lx～1500lx is most beneficial for the formation of purpurin, and the content of purpurin is the highest, also simultaneously
Improve the rate of increase of Radix Notoginseng callus successive transfer culture.
Claims (9)
1. one kind utilizes the method that Radix Notoginseng callus produces purpurin, it is characterised in that with the Radix Notoginseng plant tender stem of children, leaf,
Flower or flower pesticide as outer implant, produce callus through sterilizing, inoculation, inducing culture, callus is carried out two-stage sterilization,
Inoculation and successive transfer culture obtain passing on callus, and extract described purpurin callus from passing on；
Illumination in wherein said inducing culture controls to be 4～8 hours illumination conditions and 4～8 hours dark conditions replace into
OK；
The illumination of described successive transfer culture control be 10～15 hours illumination conditions and 10～15 hours dark conditions alternately.
A kind of method utilizing Radix Notoginseng callus to produce purpurin, it is characterised in that described more
The culture medium of injured tissue inducing culture is: MS+0.1～2mg/L 2,4-D+0.1～2mg/L BA+0.002～1mg/L TDZ+
30～60g/L sucrose；Or MS+0.1～2mg/L NAA+0.1～2mg/L BA+0.002～1mg/L TDZ+30～60g/L sugarcane
Sugar.
A kind of method utilizing Radix Notoginseng callus to produce purpurin, it is characterised in that described more
The culture medium of injured tissue successive transfer culture is: MS+0.1～2mg/L NAA+0.1～2mg/L BA+0.002～4mg/L TDZ.
A kind of utilize Radix Notoginseng callus produce purpurin method, it is characterised in that described in lure
The intensity of illumination leading cultivation is 1500 2000lx.
A kind of utilize Radix Notoginseng callus produce purpurin method, it is characterised in that described in continue
The intensity of illumination of culture is 1000 1500lx.
A kind of method utilizing Radix Notoginseng callus to produce purpurin, it is characterised in that described
The size of callus is diameter 1～2cm.
A kind of method utilizing Radix Notoginseng callus to produce purpurin, it is characterised in that described purple
The extracting method of pigment includes one or more in solvent extraction, supersound extraction, supercritical fluid extraction, microwave extraction.
A kind of method utilizing Radix Notoginseng callus to produce purpurin, it is characterised in that also include
Purification process to described purpurin.
A kind of method utilizing Radix Notoginseng callus to produce purpurin, it is characterised in that described
Purification process includes extraction, recrystallization, column chromatography for separation, one or more groups in macroporous resin separation, ion exchange resin separation
Close.
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| CN102210265A (en) * | 2010-04-02 | 2011-10-12 | 上海天龙药业有限公司 | Method for cultivating adventitious roots of pseudo-ginseng |
| CN104686340A (en) * | 2015-02-22 | 2015-06-10 | 杨业容 | Method for establishing cell suspension culture system of gynura divaricate (L.) DC. |
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